This invention relates to a method for producing L-valine by fermentation, particularly, a DNA coding for acetohydroxy acid synthase isozyme III which is free from feedback inhibition by L-valine, a microorganism which harbors the DNA, and a method for producing L-valine using the bacterium.
In the past, L-valine has been produced by a method of fermentation primarily using a microorganism belonging to the genus Brevibacterium, Corynebacterium or Serratia or a mutant thereof which produces L-valine or L-leucine (Amino acid fermentation, JAPAN SCIENTIFIC SOCIETY""S PRESS, pp.397-422, 1986). Although the conventional methods have considerably enhanced the productivity of these amino acids, the development of a more efficient, cost-effective technique is required in order to meet increasing demand for L-valine and L-leucine in the future.
As bacteria other than above-mentioned bacteria used for producion of L-valine, it is exemplified by L-valine producer belonging to the genus Escherichia which requires lipoic acid for growth and/or which is deficient in H+-ATPase activity, and a bacterium belonging to the genus Escherichia which has preceding charasteristics and which is introduced an ilvGMEDA operon expressing ilvG, ilvM, ilvE and ilvD genes and not expressing threonine deaminase (WO96/06926).
The final step of L-valine biosynthesis is carried out by a group of ilvGMEDA operon-encoded enzymes. The ilvGMEDA operon includes each of ilvG, ilvM, ilvE, ilvD and ilvA genes, which encodes a large subunit and a small subunit of isozyme II of acetohydroxy-acid synthase, transaminase, dihydroxy-acid dehydratase and threonine deaminase, respectively. Of these enzymes, acetohydroxy-acid synthase, transaminase and dihydroxy-acid dehydratase catalyze the synthetic pathways from pyruvic acid to L-valine and from 2-ketobutyric acid to L-isoleucine, and threonine deaminase catalyzes the deamination from L-threonine to 2-ketobutyric acid, which is a rate-limiting step of L-isoleucine biosynthesis. Incidentally, the expression of ilvGMEDA operon suffers control (attenuation) by L-valine and/or L-isoleucine and/or L-leucine.
As acetohydroxy acid synthase concerning L-valine biosynthesis, isozyme III (hereinafter, also referred to as AHAS III) is known, apart from isozyme II (hereinafter, also referred to as AHAS II). AHAS III is coded by ilvIH operon which consists of ilvI coding for a large subunit (catalytic subunit) and ilvH coding for a small subunit (control subunit). AHAS III suffers feedback inhibition by L-valine.
Incidentally, it has been reported that the mutant ilvH gene cloned from the mutant Escherichia coli resistant to L-valine had an amino acid substitution of 14gly with asp (Vyazmensky, M. et al., Biochemistry, 35, 10339-10346 (1996)). Further, ilvH612 has been known as the AHAS III mutation (De Felice et al., J. Bacteriol., 120, 1058-1067(1974)). The ilvH gene in the ilvIH operon of Esherichia coli MI262 (Guardiola et al., J. Bacteriol., 120, 536-538 (1974); De Felice et al., J. Bacteriol., 120, 1068-1077(1974)) contains the ilvH612 double mutation by which 29Asn is substituted with Lys and 92Gln is substituted with a termination codon(TAG), respectively.
As described above, a DNA coding for AHAS II has been utilized for breeding of L-valine producer, however, for AHAS III no case has been reported.
The object of the present invention, in view of the aforementioned points, is to provide a DNA coding for AHAS III which is free from a feedback inhibition by L-valine, a microorganism which harbors the DNA, and a method for producing L-valine using the bacterium.
As a result of diligent investigation in order to achieve the object described above, the present inventors found that L-valine productivity is increased when a DNA coding for valine resistant AHAS III isolated from an L-valine resistant mutant is introduced into Escherichia coli. Thus the present invention has been completed.
That is, aspects of the present invention are as follows:
(1) A DNA coding for a small subunit of acetohydroxy acid synthase isozyme III originating from Escherichia coli which has a mutation to replace an amino acid residue corresponding to serine residue at the amino acid number 17 with another amino acid residue in SEQ ID NO: 2, or both of a mutation to replace an amino acid residue corresponding to serine residue at the amino acid number 17 and a mutation to replace an amino acid residue corresponding to glycine residue at the amino acid number 14 with another amino acid residue in SEQ ID NO: 2;
(2) The DNA of (1), wherein the mutation of the amino acid residue corresponding to serine residue at the amino acid number 17 is replacement of the serine residue with phenylalanie residue and the mutation of the amino acid residue corresponding to glycine residue at the amino acid number 14 is replacement of the glycine residue with aspartic acid residue;
(3) A DNA coding for acetohydroxy acid synthase isozyme III originating from Escherichia coli which is free from an inhibition by L-valine and has an activity to catalyze two reactions to generate xcex1-acetolactate from pyruvate and xcex1-aceto-xcex1-hydroxybutyrate from xcex1-ketobutyrate and pyruvate;
(4) The DNA of (3), wherein the DNA codes for a large subunit and a small subunit of acetohydroxy acid synthase isozyme III, the small subunit having a mutation to replace an amino acid residue corresponding to serine residue at the amino acid number 17 with another amino acid residue, or a mutation to replace an amino acid residue corresponding to asparagine residue at the amino acid number 29 with another amino acid residue, or a mutation to delete a C-terminal region from the amino acid number 91 downwards, in SEQ ID NO: 2, or a combination of two or more mutations selected from the group consisting of aforementioned mutations and a mutation to replace an amino acid residue corresponding to glycine residue at the amino acid number 14 with another amino acid residue in SEQ ID NO: 2.
(5) The DNA of (4), wherein the mutation of the amino acid residue corresponding to serine residue at the amino acid number 17 is replacement of the serine residue with phenylalanine residue, the mutation of the amino acid residue corresponding to asparagine residue at the amino acid number 29 is replacement of the asparagine residue with lysine residue or tyrosine residue, and the mutation of the amino acid residue corresponding to glycine residue at the amino acid number 14 is replacement of the glycine residue with aspartic acid residue.
(6) A bacterium which harbors the DNA according to claims 1 or 3 on chromosomal DNA or plasmid in the bacterium and has an ability to produce L-valine;
(7) The bacterium of (6), wherein expression of the DNA is enhanced;
(8) The bacterium of (7), wherein the expression is enhanced by locating the DNA under the control of a potent promoter or amplifying a copy number of the DNA;
(9) A method for producing L-valine comprising the steps of cultivating the bacterium according to claim 6 in a culture medium, producing and accumulating L-valine in the culture medium, and collecting L-valine from the culture medium.
The present invention will be explained in detail below.
The first DNA of the present invention is a DNA encoding a small subunit of AHAS III which exhibits acetohydroxy synthase activity without suffering a feedback inhibition by L-valine along with a large subunit. Acetohydroxy synthase activity herein refers to an activity to catalyze two reactions to generate xcex1-acetolactate from pyruvate, and xcex1-aceto-xcex1-hydroxybutyrate from xcex1-ketobutyrate and pyruvate. AHAS III small subunit of Escherichia coli has an amino acid sequence depicted in SEQ ID NO: 2 in Sequence Listing.
Aforementioned mutation is selected from a mutation to replace an amino acid residue corresponding to serine residue at the amino acid number 17 with another amino acid residue in SEQ ID NO: 2, or both of a mutation to replace an amino acid residue corresponding to serine residue at the amino acid number 17 and a mutation to replace an amino acid residue corresponding to glycine residue at the amino acid number 14 with another amino acid residue in SEQ ID NO: 2. As the mutation, for the amino acid residue corresponding to serine residue at the amino acid number 17 it is preferably exemplified by replacement of the serine residue with phenylalanie residue, and for the amino acid residue corresponding to glycine residue at the amino acid number 14 it is preferably exemplified by replacement of the glycine residue with aspartic acid residue.
The second DNA of the present invention is a DNA coding for AHAS III which is free from a inhibition by L-valine and has an activity to catalyze two reactions to generate xcex1-acetolactate from pyruvate and xcex1-aceto-xcex1-hydroxybutyrate from xcex1-ketobutyrate and pyruvate. The DNA encode the large subunit and the small subunit of AHAS III, simultaneously.
The small subunit has a mutation to replace an amino acid residue corresponding to serine residue at the amino acid number 17 with another amino acid residue or a mutation to replace an amino acid residue corresponding to asparagine residue at the amino acid number 29 with another amino acid residue or a mutation to delete a C-terminal region from the amino acid number 91 downwards, in SEQ ID NO: 2, or a combination of two or more mutations selected from the group consisting of aforementioned mutations and a mutation to replace an amino acid residue corresponding to glycine residue at the amino acid number 14 with another amino acid residue in SEQ ID NO: 2. The small subunits of AHAS III which have these mutations also hereafter referred to as mutant small subunit of AHAS III. As the mutation, for the amino acid residue corresponding to serine residue at the amino acid number 17 is preferably exemplified by replacement of the serine residue with phenylalanine residue, and for the amino acid residue corresponding to asparagine residue at the amino acid number 29 it is exemplified by replacement of the asparagine residue with lysine or tyrosine residue, and for the amino acid residue corresponding to glycine residue at the amino acid number 14 it is preferably exemplified by replacement of the glycine residue with aspartic acid residue.
The DNA of the present invention was obtained from L-valine resistant mutant of Escherichia coli, however, it may be obtained by inducing above mutation or mutations into a DNA encoding wild type AHAS III by site-directed mutagenesis. AHAS III is coded by ilvIH operon. The ilvIH operon can be obtained by, for example, amplifying the DNA fragment which is from the promoter region to 3xe2x80x2 end of ilvH gene by PCR using primers having sequences depicted in SEQ ID NOs: 3 and 4 from genomic DNA of Escherichia coli as a template. The nucleotide sequence of ilvIH operon has been known (Genbank/EMBL/DDBJ accession X55034). The nucleotide sequence of coding region of ilvH is illustrated in SEQ ID NO: 1.
The mutant small subunit of AHAS III coded by the DNA of the present invention may have an amino acid sequence which includes substitution, deletion, insertion, addition, or inversion of one or several amino acids as well as aforementioned mutation, provided that the mutant small subunit exhibits acetohydroxy acid synthase activity without suffering a feedback inhibition by L-valine along with the large subunit.
A DNA, which codes for the substantially same protein as the mutant small subunit as described above, is obtained, for example, by modifying the nucleotide sequence, for example, by means of the site-directed mutagenesis method so that one or more amino acid residues at a specified site involve substitution, deletion, insertion, addition, or inversion. DNA modified as described above may be obtained by the conventionally known mutation treatment. The mutation treatment includes a method for treating DNA coding for the small subunit in vitro, for example, with hydroxylamine, and a method for treating a bacterium belonging to the genus Escherichia harboring the DNA coding for the small subunit with ultraviolet irradiation or a mutating agent such as N-methyl-Nxe2x80x2-nitro-N-nitrosoguanidine (NTG) and nitrous acid usually used for the mutation treatment.
The DNA, which codes for substantially the same protein as mutant small subunit of AHAS III, is obtained by expressing DNA having mutation as described above in multicopy in an appropriate cell, investigating the resistance to L-valine, and selecting the DNA which increase the resistance. Also, it is generally known that an amino acid sequence of a protein and a nucleotide sequence coding for it may be slightly different between strains, mutants or variants, and therefore the DNA, which codes for substantially the same protein, can be obtained from L-valine resistant species, strains, mutants and variants belonging to the genus Escherichia.
Specifically, the DNA, which codes for substantially the same protein as the mutant small subunit, can be obtained by isolating a DNA which hybridizes with DNA having, for example, a nucleotide sequence shown in SEQ ID NO: 1 in Sequence Listing under stringent conditions, and which codes for a protein having the acetohydroxy acid synthase activity, from a bacterium belonging to the genus Escherichia which is subjected to mutation treatment, or a spontaneous mutant or a variant of a bacterium belonging to the genus Escherichia. The term xe2x80x9cstringent conditionsxe2x80x9d referred to herein is a condition under which so-called specific hybrid is formed, and non-specific hybrid is not formed. It is difficult to clearly express this condition by using any numerical value. However, for example, the stringent conditions include a condition under which DNAs having high homology, for example, DNAs having homology of not less than 70% with each other are hybridized, and DNAs having homology lower than the above with each other are not hybridized.
The bacterium of the present invention harbors the first DNA or the second DNA of the present invention and has an activity to produce L-valine. The bacterium is not particularly limited so long as it has a biosynthetic pathway of L-valine which acetohydroxy acid synthase concerns with. It is exemplified by a bacterium belonging to the genus Escherichia, coryneform bacteria and the genus Serratia, preferably by the genus Escherichia. A bacterium belonging to the genus Escherichia is concretely exemplified by Escherichia coli. 
Examples of a method for introducing the DNA of the present invention into a bacterium include, for example, a method in which a bacterium is transformed with a plasmid containing the DNA of the present invention, and a method in which the DNA of the present invention is integrated into chromosomal DNA of a bacterium by homologous recombination, or the like.
It is preferable that expression of the DNA of the present invention is enhanced. The enhancement of expression is achieved by locating the DNA of the present invention under the control of a potent promoter or amplifying a copy number of the DNA. For example, lac promoter, trp promoter, trc promoter, tac promoter, PR promoter, PL promoter of lambda phage, tet promoter, amyE promoter and spac promoter are known as potent promoters. Also, it is possible to increase the copy number of the DNA of the present invention by maintaining the DNA on a multi-copy vector or introducing multiple copies of the DNA into the chromosomal DNA. The multi-copy vector is exemplified by pBR322, pTWV228, pMW119 and pUC19 or the like.
To introduce the vector containing the DNA of the present invention to a host bacterium, any known transformation methods can be employed. For instance, employable are a method of treating recipient cells with calcium chloride so as to increase the permeability of DNA, which has been reported for Escherichia coli K-12 [see Mandel, M. and Higa, A., J. Mol. Biol., 53, 159 (1970)]; and a method of preparing competent cells from cells which are at the growth phase followed by introducing the DNA thereinto, which has been reported for Bacillus subtilis [see Duncan, C. H., Wilson, G. A. and Young, F. E., Gene, 1, 153 (1977)]. In addition to these, also employable is a method of making DNA-recipient cells into the protoplast or spheroplast which can easily take up recombinant DNAs followed by introducing the recombinant DNA into the cells, which is known to be applicable to Bacillus subtilis, actinomycetes and yeasts [see Chang, S. and Choen, S. N., Molec. Gen. Genet., 168, 111 (1979); Bibb, M. J., Ward, J. M. and Hopwood, O. A., Nature, 274, 398 (1978); Hinnen, A., Hicks, J. B. and Fink, G. R., Proc. Natl. Sci., USA, 75, 1929 (1978)], or a method transformation used in embodiments of the present invention is the electric pulse method (refer to Japanese Patent Publication Laid-Open No. 2-207791).
Applicable method to introduce the DNA of the present invention into bacterial chromosomal DNA includes a method utilizing linearized DNA and that utilizing a plasmid containing a temperature-sensitive replication origin. Alternatively, the DNA of the present invention may be introduced into a bacterium from a bacterium harboring the DNA of the present invention on its chromosomal DNA by transduction.
In order to introduce multiple copies of the DNA of the present invention into the chromosomal DNA of a bacterium, the homologous recombination is carried out using a sequence whose multiple copies exist in the chromosomal DNA as targets. As sequences whose multiple copies exist in the chromosomal DNA, repetitive DNA, inverted repeats exist at the ends of a transposable element can be used. Also, as disclosed in Japanese Patent Publication Laid-Open No. 2-109985, it is possible to incorporate the DNA of the present invention into transposon, and allow it to be transferred to introduce multiple copies of the DNA into the chromosomal DNA.
The bacterium to which the DNA of the present invention is introduced may be a bacterium being acquired L-valine productivity by introduction of the DNA of the present invention as well as a bacterium inherently having L-valine productivity.
Examples of bacteria having L-valine productivity includes, for example, Escherichia coli VL1970 (U.S. Pat. No. 5,658,766). Additionally, bacteria described in W096/06926 such as L-valine producer belonging to the genus Escherichia which requires lipoic acid for growth and/or which is deficient in H+-ATPase activity, or a bacterium belonging to the genus Escherichia which is introduced an ilvGMEDA operon expressing at least ilvG, ilvM, ilvE and ilvD genes are preferably used. Since the expression of ilvGMEDA operon suffers control (attenuation) by L-valine and/or L-isoleucine and/or L-leucine, it is preferable that the region which is essential for attenuation is deleted or mutated to desensitize the repression of expression by produced L-valine. Another approach suggests the introduction of the mutations (ileS or valS) affecting aminoacyl-tRNA synthases having decreased affinity (increased the Km) for the corresponding amino acids. Further, the operon which does not express active threonine deaminase is used preferably.
Escherichia coli VL1970 containing ileS17 mutation in which attenuation is desensitized as described above has been deposited in Russian National Collection of Industrial Microorganisms (VKPM) Depositary, GNIIgenetika, (1, Dorozhny Proezd., 1, 113545, Moscow, Russia) under the accession number of VKPM B-4411.
The methods to perform, for example, hybridization, PCR, preparation of plasmid DNA, digestion and ligation of DNA, and transformation are described by Sambrook, J., Fritsche, E. F., Maniatis, T. in Molecular Cloning, Cold Spring Harbor Laboratory Press, 1.21 (1989).
The production of L-valine can be performed by culturing the bacterium having L-valine productivity in a medium, to allow L-valine to be produced and accumulated in the medium, and collecting L-valine from the medium.
In the present invention, the cultivation, the collection and purification of L-valine from the medium and the like may be performed in a manner similar to the conventional fermentation method wherein an amino acid is produced using a microorganism. A medium used for culture may be either a synthetic medium or a natural medium, so long as the medium includes a carbon source and a nitrogen source and minerals and, if necessary, appropriate amounts of nutrients which the microorganism requires for growth. The carbon source may include various carbohydrates such as glucose and sucrose, and various organic acids. Depending on the mode of assimilation of the used microorganism, alcohol including ethanol and glycerol may be used. As the nitrogen source, various ammonium salts such as ammonia and ammonium sulfate, other nitrogen compounds such as amines, a natural nitrogen source such as peptone, soybean-hydrolysate and digested fermentative microorganism are used. As minerals, potassium monophosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, calcium carbonate, and the like are used.
The cultivation is performed preferably under aerobic conditions such as a shake culture, and an aeration and stirring culture, at a temperature of 20 to 40xc2x0 C., preferably 30 to 38xc2x0 C. The pH of the culture is usually between 5 and 9, preferably between 6.5 and 7.2. The pH of the culture can be adjusted with ammonia, calcium carbonate, various acids, various bases, and buffers. Usually, a 1 to 3-day cultivation leads to the accumulation of the target L-valine in the liquid medium.
After cultivation, solids such as cells can be removed from the liquid medium by centrifugation and membrane filtration, and then the target L-valine can be collected and purified by ion-exchange, concentration and crystallization methods.